The object of the proposed research program is to study the role of cysteine residues in the different facets of hemoglobin structure and function (tetramer formation, oxygen affinity, etc.). Hemoglobins A, F and S have been chosen because of the differences which exist in the primary structure of the beta-like subunit of these molecules. The cysteine residues will be modified by reaction with p-mercuribenzoate (PMB) and mixtures of isolated subunits with PMB attached to some or all of the sulfhydryl groups will be made. Products from these mixtures which resemble hemoglobin A chromatographically and electrophoretically will be isolated and their kinetic and equilibrium properties during oxygen and carbon monoxide binding will be compared with respect to differences in primary sequence of the beta-subunit. In addition, the effect of cysteine modification on the kinetics of methemoglobin formation from oxy-hemoglobin will be examined. The cysteine residues will also be used as sites for spin label probes which will be used to monitor by electron spin resonance, structural changes which take place within the different hemoglobin molecules during reversible oxygen binding. The information obtained from these studies should help elucidate the nature of the structural differences between these three hemoglobins and therefore provide further information concerning their functional differences, such as the involvement of hemoglobin S in sickle cell anemia.